The cells in culture adopt a random spatial organization in which the positioning of the cells is unforeseeable. The cells are permanently pulled and pushed. They thus move according to the fluctuations of the intercellular interactions. No tool making it possible to control the positions and the shapes of the cells within these multicellular arrangements is available.
There are some tools to isolate cells from each others and control their shapes: the micro-patterning. Accordingly, adhesive patterns can be prepared by grafting extracellular matrix proteins on a solid support. Those proteins induce cellular adherence and promote cell attachment and flattening on the solid support. Consequently, cells can be confined in squares, rounds, triangles or more complex geometries, their size corresponding to the size of a cell. Cells have then all the same shape and even can adopt very reproducible internal organizations. Suitable micro-patterns have been disclosed in the PCT application WO 2005/026313. However, cells thus constrained are isolated and are not in direct contact with other cells. Accordingly, each cell is isolated from the other cells on an adhesive pattern by surrounding cytophobic regions in order to avoid any contact between cells immobilized on the support. The size of the adhesive pattern is such as one single cell could spread. This is the major drawback of this technology.
Indeed, the biologic mechanisms controlling the mechanic and functional coherence of a tissue by regulating the size, shape and position of cells do not only depend on the cell anchorage with extracellular matrix but also on the attachments of cells to the neighbouring cells. The cell-cell interactions, in addition to their mechanical role, also contribute to biochemical signalization pathways that regulate cellular activities as important as the proliferation, the differentiation and the apoptosis. In order to reproduce in a cell culture the conditions of a cell in a tissue, two types of contact are therefore needed: contacts with the extracellular matrix and contacts between cells.
The simplest solution to reach the shape control of a group of several cells from the one of an individual cell is to use a larger micro-pattern (Nelson and Chen, 2002, FEBS Letters, 514, 238-242).
However, the micro-patterning technology reaches its limit in this context. Indeed, when two cells are constrained on a same pattern, they can establish the two types of contact (cell-matrix with the adhesive pattern and cell-cell with the neighbouring cells), but they can also move and for instance take the place of the other cell. Hence, they tend to move around each other. They do not adopt a mechanical equilibrium allowing them to stabilize in one stationary configuration. Scientific articles were devoted to the movement of a pair of cells on micro-patterns (Brangwynne et al, 2000, In Vitro Cell Dev Biol Anim, 36, 563-5; Huang et al, 2005, Cell Motil Cytoskeleton, 61, 201-213). In addition, the cells position, the position and size of cell-cell contact zone are not controlled so as the polarity and the internal organization (Nelson and Chen, 2002). In Huang et al, 2005, the authors observed that two NIH 3T3 cells on a square pattern did not move around each other and formed a straight cell-cell boundary stretching diagonally between opposite corners of the square.
Nelson et al develops another system to control the position of cells and the cell-cell contact (Nelson and Chen, 2002, supra; Nelson and Chen, 2003, J Cell Science, 116, 3571-3581). Cells are constrained in agarose micro-wells of about 10 μm of depth. Micro-wells have a bow-tie shape. Agarose prevents protein adsorption and cell adhesion. When two cells are present in a micro-well, each cell is located in one of the two triangular forms of the bow-tie shape. The micro-wells depth and their agarose composition allow the constraint of the cells into the triangular forms and the cells cannot step over to move into the other triangular half. However, in such bow-tie shaped micro-wells, the structure of actin does not seem to be well controlled. In addition, the intercellular contact zone is narrowed to the bottle neck between the two triangular forms and the bottle neck has to be narrow for preventing cells to move into the other triangular half. In addition, the cells are not in a mechanical equilibrium. Indeed, the cell position is restrained by the form of the bow-tie shaped micro-wells. Accordingly, the cell configuration is not based on mechanical interaction between cells. Therefore, the conformation is artefactitious and does not reproduce the physiological conditions of cells in a tissue.
Accordingly, there is a strong need of methods and devices to obtain multicellular arrangements in stable, stationary and reproducible spatial configuration, and optionally with controlled internal cell organization.